Type II restriction enzymes (REases) are indispensible tools of modern medical research. It has long been a goal of REase manufacturers to be able to offer programmable specificity enzymes, where the sequence recognized and resulting cutting activity can be precisely directed to any desired point in a DNA, as this will offer opportunity for improvement in many applications, from DNA sequencing to gene therapy. We have identified a new family of Type II endonucleases that are amenable for the first time to the rational engineering of new DNA binding and cleavage specificities. To commercialize potentially thousands of new enzymes, we propose a structure-based approach, using crystallographic information to identify specificity determinants, which can then be rationally mutated to generate new nucleases with programmable specificities. In phase I of this application, we will prepare large amounts of several MmeI family enzymes, with the goal of having in hand well-diffracting crystals for two MmeI-family enzymes. In preliminary studies we have obtained well-diffracting cocrystals of MmeI that are highly suitable for structure determination. In Phase II, we will first determine structures for two MmeI family enzymes bound to DNA and then, as part of aim 2, use that information together with structure-based amino acid sequence alignments to generate a code of position-specific amino acids for the engineering of nucleases with programmable specificities. In aim 3, we will generate the potentially thousands of new specificity enzymes using site-directed mutagenesis protocols and, in aim 4, refine our understanding of specificity within this novel family of enzymes through structures of select mutants. An important application of MmeI-like enzymes is in technologies such as Serial Analysis of Gene Expression (SAGE) and paired-end sequence reads in next-generation DNA sequencing methods. Thus, as part of aim 5, we will use the structural information to engineer enzymes with extended reach between the recognition and cleavage sites for improving the quality of SAGE data and for speeding the assembly of genomes in DNA sequencing methods. Engineered Mme-I-like enzymes also offer the potential for targeted therapeutic use with minimal off target cleavage and toxicity. In aim 6, we will use our structural information to generate rare cutting MmeI-like endonucleases for therapeutic use.